TGF-beta signaling in pancreatic cell differentiation

Abstract

Abstract Introduction: TGF-beta signaling has been implicated in pancreatic cancer due to the prevalence of smad4 mutations. An established model for pancreatic endocrine differentiation is exendin-4 (glucagon-like peptide-1 analog) treatment of AR42J cells (rat pancreatic carcinoma cell line). We studied TGF-beta superfamily signaling and downstream intracellular effectors (smad proteins) implicated in many developmental processes. Here, we found a novel interdependence between GLP-1 and TGF-beta signaling essential for endocrine differentiation. Methods: AR42J cells were cultured with exendin-4 at serial doses for three days. For inhibitory studies, morpholino antisense and missense controls were added to culture media. Other bioactivity studies were performed using either TGF-beta ligands or else a pan-specific TGF-beta neutralizing antibody. Cell differentiation was analyzed by semi-quantitative PCR, as well as immunocytochemistry (IFCC). Results: Smad2 and smad3 mRNA levels were respectively decreased and increased in response to exendin-4 treatment. Further, smad2 antisense or smad2/smad3 double antisense prevented AR42J cells from becoming insulin-positive. Surprisingly, smad3 antisense alone led to a 6.5-fold enhancement of insulin mRNA levels and a doubling of insulin-positive cells by IFCC. The addition of TGF-beta1 ligand greatly upregulated smad3 mRNA and led to a dramatic elevation in insulin expression. Interestingly, TGF-beta smads and insulin-positive differentiation were completely blocked by TGF-beta antibodies, or TGF-beta receptor type I antisense (Alk-1 and Alk-5). Conclusions: TGF-Beta isoform signaling represents a key control point in regulating the level of differentiation of pancreatic AR42J cancer cells.